Volume 10, Issue 10, Pages 1334-1348 (October 2017) Lamin-like Proteins Negatively Regulate Plant Immunity through NAC WITH TRANSMEMBRANE MOTIF1-LIKE9 and NONEXPRESSOR OF PR GENES1 in Arabidopsis thaliana  Tongtong Guo, Xuegao Mao, Hui Zhang, Yu Zhang, Mengdi Fu, Zhenfei Sun, Peng Kuai, Yonggen Lou, Yuda Fang  Molecular Plant  Volume 10, Issue 10, Pages 1334-1348 (October 2017) DOI: 10.1016/j.molp.2017.09.008 Copyright © 2017 The Author Terms and Conditions

Figure 1 CRWN1 and CRWN2 Negatively Regulate the Resistance to Psm ES4326 and Expression of PR1. (A) Leaves of wild-type (WT), crwn1 crwn2 double mutant, and crwn1 and crwn2 single mutants were infected with Psm ES4326 (OD600 = 0.002). Disease symptoms at 3 days post inoculation (dpi) are shown. (B) Growths of Psm ES4326 in infected leaves at 0 and 3 dpi. Data are shown as mean ± SEM (n = 8). Significant differences between mutants and WT at 3 dpi are calculated by one-way ANOVA followed by Bonferroni's multiple comparison test. *P < 0.05, ****P < 0.0001; ns, no significant difference. The bacterial titer at 0 dpi was not significantly different among all four genotypes. The experiment was repeated three times with similar results. c.f.u., colony-forming units. (C) The transcript levels of PR1 were monitored by real-time PCR in crwn1 crwn2 double mutant and WT 2 days after incubation with Psm ES4326 (OD600 = 0.002) or 10mM MgCl2 as a control. The relative transcript levels of PR1 were normalized to the transcript of ACTIN2. Data are presented as mean ± SEM (n = 3 biological replicates). Statistically significant differences between crwn1 crwn2 and WT are analyzed by multiple t tests. **P < 0.01, ***P < 0.001. Molecular Plant 2017 10, 1334-1348DOI: (10.1016/j.molp.2017.09.008) Copyright © 2017 The Author Terms and Conditions

Figure 2 The Transcription of CRWN1 Is Induced by Pathogens and SA. Leaves of 3- to 4-week-old WT plants were infiltrated with bacterial suspensions of Pst DC3000 (A) or Psm ES4326 (B) (OD600 = 0.002). Leaves of 4-week-old WT plants were treated with 0.5 mM SA (C). Leaves were harvested at the indicated time points in (A), (B), and (C) after these treatments. The relative transcript levels of CRWN1 were normalized to the transcript level of ACTIN2. Data are presented as mean ± SEM (n = 3 biological replicates). Statistically significant differences from 0 h after these treatments were analyzed by one-way ANOVA followed by Bonferroni's multiple comparisons test. *P < 0.05, ***P < 0.001, ****P < 0.0001. Molecular Plant 2017 10, 1334-1348DOI: (10.1016/j.molp.2017.09.008) Copyright © 2017 The Author Terms and Conditions

Figure 3 Pathogen and SA Induce the Proteasome-Mediated Degradation of CRWN1. (A–D) Leaves of 3- to 4-week-old Pro35S:CRWN1-YFP (A) and ProCRWN1:CRWN1-YFP (C) plants were infiltrated with Psm ES4326 (OD600 = 0.002) for the indicated hours. Pro35S:CRWN1-YFP (B) and ProCRWN1:CRWN1-YFP (D) seedlings were treated with 1 mM SA for indicated time periods and 1 mM SA combined with MG132 for 30 min. Total proteins were extracted and CRWN1-YFP was analyzed by western blot using an anti-GFP antibody. Detection of constitutively expressed ACTIN serves as a loading control. (E) Leaves of Pro35S:CRWN1-YFP/ProHTR4:HTR4-mCherry plants were treated with 1 mM SA or 0.1% ethanol, which was used as a control. Leaves were observed under fluorescence microscopy 30 min after treatment. Scale bar, 10 μm. (F) Total protein was extracted from Pro35S:CRWN1-YFP in a buffer supporting proteolytic activity. Extracts were incubated at room temperature for indicated time periods. CRWN1-YFP was detected by western blot using an anti-GFP antibody. Detection of constitutively expressed ACTIN served as a loading control. (G) Total protein was extracted from Pro35S:CRWN1-YFP plants in the same proteolytic buffer as in (F), then incubated at room temperature for 2 h with addition of 2% DMSO, 40 mM MG132, 40 mM MG115, 4 mM PMSF, or cocktail. CRWN1-YFP was detected by western blot using an anti-GFP antibody with CBB as a loading control. (H) Leaves of Pro35S:CRWN1-YFP plants were treated with 0.1% ethanol, 1 mM SA, 1 mM SA combined with MG115 or MG132 for 90 min, then observed under fluorescence microscopy. Scale bar, 10 μm. (I) Quantitative analysis of relative YFP fluorescence in (H). Data are presented as mean ± SEM (n = 10). Statistically significant differences from SA treatment are analyzed by one-way ANOVA followed by Bonferroni's multiple comparisons test. ****P < 0.0001. Molecular Plant 2017 10, 1334-1348DOI: (10.1016/j.molp.2017.09.008) Copyright © 2017 The Author Terms and Conditions

Figure 4 CRWN1 Interacts with NTL9. (A) Yeast two-hybrid assays show that CRWN1 interacts with NTL9 through their C-terminal fragments. Co-transformed yeast colonies were mixed with 50 μl of water respectively diluted by 1:10, 1:100, and 1:1000, and dropped onto the selective SD medium minus His, Trp, and Leu supplemented with 3 mM 3-amino-1,2,4-triazole (3-AT), or SD medium minus Trp and Leu as a control. CRWN1C (aa 799–1132), NTL9 (full length, aa 1–512), NTL9N (aa 1–246), NTL9C (aa 247–512), NTL9C1 (aa 247–367), NTL9C2 (aa 368–512). (B) MBP pull-down assays show the interaction between CRWN1C and NTL9. The arrows indicate aim bands, from top to bottom: MBP-CRWN1C, MBP, GST-NTL9, GST, and GST-NTL9. (C) Luciferase complementation imaging assays show the interaction between CRWN1 and NTL9. Luc activity was detected for CRWN1-NLuc/CLuc-NTL9, with SNI1-NLuc/CLuc-NTL9 as a positive control and CRWN1-NLuc/CLuc vector or NLuc vector/CLuc-NTL9 as the negative controls. (D) Annotation of the distribution in (C). Molecular Plant 2017 10, 1334-1348DOI: (10.1016/j.molp.2017.09.008) Copyright © 2017 The Author Terms and Conditions

Figure 5 CRWN1 Suppresses the Expression of PR1 by Enhancing the Binding of NTL9 to PR1 Promoter. (A) The relative transcript levels of PR1, ICS1, EDS1, and PAD4 in WT, ntl9, crwn1 crwn2, and ntl9 crwn1 crwn2 lines examined by real-time PCR. The relative fold changes were normalized to the transcript of ACTIN2. Data are presented as mean ± SEM (n = 3 biological replicates). Statistical significance was analyzed by one-way ANOVA followed by Bonferroni's multiple comparisons test. Means with different letters are significantly different from each other. (B) Transient luciferase expression assays show that CRWN1 enhances the inhibition of NTL9 on the activity of PR1 promoter. (C) Dual-LUC assays performed in tobacco. The relative LUC activities of the indicated genotypes were measured and normalized to the REN activity as shown (LUC/REN). Data are presented as mean ± SEM (n = 3). Statistical significance was analyzed by one-way ANOVA followed by Bonferroni's multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001. The experiment was repeated three times with similar results. (D) EMSAs show MBP-NTL9 binds to the E0-1-1 element of PR1 promoter. Biotin-labeled 5 pM E0-1-1 element was added in all lanes, and unlabeled E0-1-1 element was added in lane 3. Lane 2, 1 μg MBP-NTL9; lane 3, 1 μg MBP-NTL9 + 200 pM unlabled E0-1-1 element; lane 4, 1 μg MBP. (E) EMSAs show that CRWN1C enhances the binding of NTL9 to the E0-1-1 element of PR1 promoter. Recombinant proteins and biotin-labeled 5 pM E0-1-1 element were added in all lanes. Lane 1, 1 μg MBP-NTL9; lane 2, 1 μg MBP; lane 3, 1 μg MBP-NTL9 + 1 μg MBP; lane 4, 1 μg MBP- CRWN1C; lane 5, 1 μg MBP-NTL9 + 1 μg MBP- CRWN1C; lane 6, 1 μg MBP-NTL9 + 2 μg MBP- CRWN1C; lane 7, 1 μg MBP-NTL9 + 3 μg MBP- CRWN1C; lane 8, 1 μg MBP-NTL9 + 4 μg MBP-CRWN1C; lane 9, 1 μg MBP-NTL9 + 6 μg MBP-CRWN1C. (F) ChIP-PCR shows interaction between NTL9 and PR1 promoter decreases in crwn1 crwn2. ChIP-PCR was performed in NTL9 OE/WT and NTL9 OE/crwn1 crwn2 lines using a GFP antibody. Samples without addition of GFP antibody served as the negative controls. ACTIN was used as internal controls for the ChIP experiments. ChIP-PCR results were quantified by normalization of GFP-IP signal with the corresponding input signal (IP[PR1/ACTIN]/input[PR1/ACTIN]). Data are presented as mean ± SEM (n = 3 biological repeats). Statistical significance was analyzed by one-way ANOVA followed by Tukey's multiple comparisons test. ****P < 0.0001. Molecular Plant 2017 10, 1334-1348DOI: (10.1016/j.molp.2017.09.008) Copyright © 2017 The Author Terms and Conditions

Figure 6 Whole-Plant and Pathogen Resistance Phenotypes of crwn1 crwn2 and crwn1 crwn2 npr1 Mutants. (A) Whole-plant phenotype of crwn1 crwn2 was compromised by npr1. Three-week-old WT, crwn1 crwn2, npr1, and crwn1 crwn2 npr1 plants grown in long-day photoperiods are shown. (B) Leaves of crwn1 crwn2, npr1, WT, and crwn1 crwn2 npr1 were infected with Psm ES4326 (OD600 = 0.002). The disease symptoms in leaves are depicted at 3 dpi. (C) Bacterial growths in infected leaves at 0 and 3 dpi. Data are shown as mean ± SEM (n = 8). Significant difference was calculated by one-way ANOVA followed by Bonferroni's multiple comparisons test. Means with different letters are significantly different from each other. The bacterial titer on 0 dpi was not significantly different among all four genotypes. The experiment was repeated three times with similar results. (D) The transcript levels of PR1 were monitored by real-time PCR in WT, crwn1 crwn2, npr1, and crwn1 crwn2 npr1 lines at 0, 12, 24, and 48 hours after incubation with Psm ES4326 (OD600 = 0.002). The relative transcript levels of PR1 were normalized to the transcript of ACTIN2. Data are presented as mean ± SEM (n = 3 biological replicates). Statistical significance was analyzed by two-way ANOVA followed by Tukey's multiple comparisons test. Means with the same letter are not significantly different from each other. Molecular Plant 2017 10, 1334-1348DOI: (10.1016/j.molp.2017.09.008) Copyright © 2017 The Author Terms and Conditions

Figure 7 A Model for the Role of CRWN1 in PR1 Expression Induced by Pathogen and SA. Pathogen and SA induce the degradation of CRWN1, which interacts with NTL9 and SNI1 to repress the transcription of PR1. Npr1 genetically suppresses the function of CRWN1, while sni1 was identified as a genetic suppressor of npr1. The complexes CRWN1/NTL9/SNI1 and NPR1/TGAs act antagonistically and dynamically to regulate the transcription of PR1 gene. Molecular Plant 2017 10, 1334-1348DOI: (10.1016/j.molp.2017.09.008) Copyright © 2017 The Author Terms and Conditions